The millisecond timescales inherent to action potentials in neurons, calcium waves in cardiac tissue, and conformational changes in proteins demand imaging instrumentation with sub-millisecond time resolution for their study. The ability to resolve the fastest biological processes over a large field of view will enable new directions i research and a better understanding of many electro-biochemical and biochemical processes. Thanks to fluorescent probes, many of these events are observable using optical microscopy. However, their weak fluorescent emission requires that imaging devices use long integration times to collect enough photons to generate images with high SNR, which result in low image acquisition speed. Technologies such as the electron-multiplier charge coupled device (EMCCD) offer electronic gain to compensate for the small number of photons detected during a shorter integration time, but the serial pixel readout strategy ultimately limits the full-frame (512x512 pixels) rate to less than 100 Hz. Photomultiplier tubes (PMTs) offer high gain and high- speed readout, but are typically manufactured in single element detector formats. For these reasons, fluorescence microscopy has been unable to resolve millisecond transients with full field, diffraction-limited spatial resolution. The goal of this proposal is to develop fluorescence microscopy instrumentation for high-speed imaging applications in biology. The introduction of a confocal fluorescence microscope capable of kilohertz frame rates with sufficient sensitivity and resolution to resolve the millisecond-timescale dynamics in living cells and tissues will enable new discoveries in all areas of biology. To accomplish this goal, we propose to employ techniques from the field of radiofrequency (RF) communications to multiplex the fluorescence excitation and emission of samples such that many pixels can be imaged simultaneously using a single PMT. We have deemed this technology Fluorescence Imaging using Radiofrequency-multiplexed Excitation, or FIRE. While this technology should find application in all areas of biology, we envision this system to make the most impact by enabling new science and aiding the development of insight into the operation of the brain and heart, where fluorescence-based calcium and voltage imaging speed are at a premium (e.g, action potential = 1 ms).In the first year, we will further develop our prototype, in order to improve bot the temporal and spatial resolutions. Our preliminary data from experiments imaging fixed adherent cells using one-photon excitation demonstrates the feasibility of this technique, and we will extend these experiments to live cell calcium imaging. In the second year, we will extend the high-speed one-photon imaging concept to two-photon excitation fluorescence imaging, using calcium imaging of neuronal network activity as proof-of-principle. During the third year, we will demonstrate the FIRE's advances by demonstrating its utility in imaging neuronal activity in the brain of urethane-anesthetized mice with unprecedented time resolution.